PROJECT SUMMARY Since the unexpected emergence of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in 2013, the ongoing outbreaks of MERS in the Middle East and the potential for global transmission of MERS, exemplified by an outbreak in South Korea in 2015, have underscored the urgent need for effective preventive and therapeutic measures against this highly virulent coronavirus. MERS-CoV expresses two polyproteins that undergo proteolytic processing by two virus-encoded proteases, a 3C-like protease (3CLpro) and a papain-like protease, to generate functionally active proteins. MERS-CoV 3CLpro processes the majority of the cleavage sites on the polyproteins and is essential for viral replication, making it an attractive therapeutic target. A series of potent dipeptidyl inhibitors of the 3CLpro of coronaviruses including MERS-CoV and infectious peritonitis coronavirus (FIPV), a highly virulent feline coronavirus, have been generated. Using FIPV as a model, it was demonstrated that the lead compound for FIPV reverses the progression of fatal FIP in experimentally or naturally infected cats. Since FIP disease progression is quite rapid and its pathogenesis primarily immune- mediated, features shared by MERS-CoV, it was hypothesized that a viral protease inhibitor could reverse the pathogenesis of MERS-CoV in affected hosts. Using a structure-guided approach, the anti-FIPV compound was structurally modified resulting in the identification of piperidine-derived lead compounds that were found to be highly effective against MERS-CoV. Thus, the primary goal of this R01 application is the identification of an in-vivo validated MERS-CoV preclinical candidate by conducting an array of basic and applied studies. Four aims are proposed to achieve this objective. Specific Aim 1. Optimize the piperidine-derived lead series of MERS-CoV 3CLpro inhibitors by iterative medicinal chemistry and structure-based drug design. Specific Aim 2. Conduct in vitro efficacy, biochemical, mechanistic, structural, spectroscopic, and computational studies to prioritize analogs, elucidate the mechanism of action, and accelerate the optimization process. Specific Aim 3. Evaluate the physicochemical properties, ADMET, PK, and oral bioavailability of optimized leads. Specific Aim 4. Determine in vivo efficacy of optimized leads in mouse models of MERS-CoV infection. The ultimate long term goal of this program is the development of antiviral therapeutics for MERS by advancing a drug candidate through the stage of filing for an investigational new drug (IND) application.